The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO₂ NPs were used to compare the effects. Exposure to 32?μg/mL ZnO NPs (p?2 NPs (p?>?0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO₄ induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO₂ NP exposure (p?>?0.05). The presence of 250?nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p?p?>?0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p?>?0.05) except neutral red uptake assay (p?相似文献   

Genotoxicity and cytotoxicity of ZnO and Al2O3 nanoparticles

Abstract

Objectives: Metal oxide nanoparticles (ZnO-NPs and Al₂O₃-NPs) are used in many fields, including consumer products and biomedical applications. As a result, exposure to these NPs is highly frequent, however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms are available. For this reason, we studied cytotoxic and genotoxic effects of ZnO-NPs and Al₂O₃-NPs on human peripheral blood lymphocytes.

Materials and methods: We obtained our goals by using MTT assay, Annexin V-FITC flow cytometry, and alkaline, neural and pH 12.1 versions of comet assay.

Results: Exposure of lymphocytes to both NPs for 24?h slightly decreased viability of lymphocytes at ≥0.5?mM. For the first time, we revealed using the comet assays that both ZnO-NPs and Al₂O₃-NPs caused a concentration-dependent increase of DNA single-strand breaks, but not alkali-labile sites. Treatment with DNA glycosylases showed that the NPs induced oxidative DNA damage. DNA damage caused by both nanoparticles at 0.05?mM was removed within 120?min, however lymphocytes did not repair DNA damage induced by 0.5?mM NPs. Studied nanoparticles did not induce apoptosis in lymphocytes.

Conclusion: Our results suggest that ZnO-NPs and Al₂O₃-NPs at concentration up to 0.5?mM did not exhibit cytotoxic effect but may exert genotoxic effect on lymphocytes, at least partially by the generation of oxidative DNA damage and strand breaks.     

Toxicity of TiO2 nanoparticles on soil nitrification at environmentally relevant concentrations: Lack of classical dose–response relationships

Titanium-dioxide nanoparticles (TiO₂-NPs) are increasingly released in agricultural soils through, e.g. biosolids, irrigation or nanoagrochemicals. Soils are submitted to a wide range of concentrations of TiO₂-NPs depending on the type of exposure. However, most studies have assessed the effects of unrealistically high concentrations, and the dose–response relationships are not well characterized for soil microbial communities. Here, using soil microcosms, we assessed the impact of TiO₂-NPs at concentrations ranging from 0.05 to 500?mg kg^?1?dry-soil, on the activity and abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB), and nitrite-oxidizing bacteria (Nitrobacter and Nitrospira). In addition, aggregation and oxidative potential of TiO₂-NPs were measured in the spiking suspensions, as they can be important drivers of TiO₂-NPs toxicity. After 90?days of exposure, non-classical dose–response relationships were observed for nitrifier abundance or activity, making threshold concentrations impossible to compute. Indeed, AOA abundance was reduced by 40% by TiO₂-NPs whatever the concentration, while Nitrospira was never affected. Moreover, AOB and Nitrobacter abundances were decreased mainly at intermediate concentrations nitrification was reduced by 25% at the lowest (0.05?mg?kg^?1) and the highest (100 and 500?mg?kg^?1) TiO₂-NPs concentrations. Path analyses indicated that TiO₂-NPs affected nitrification through an effect on the specific activity of nitrifiers, in addition to indirect effects on nitrifier abundances. Altogether these results point out the need to include very low concentrations of NPs in soil toxicological studies, and the lack of relevance of classical dose–response tests and ecotoxicological dose metrics (EC50, IC50…) for TiO₂-NPs impact on soil microorganisms.     

Comparison of the effects of MnO2-NPs and MnO2-MPs on mitochondrial complexes in different organs

Today, nanoparticles (NPs) have been widely used in various fields. Manganese oxide nanoparticles have attracted a lot of attention due to many applications. One of the major concerns regarding the widespread use of various NPs is the exposure and accumulation in human organs and finally toxicity. The generation of reactive oxygen species (ROS) by mitochondria is one of the most important mechanisms of toxicity suggested by published studies induced by other NPs. However, limited studies have been conducted on the mechanism of toxicity of MnO₂-NPs and MnO₂-microparticles (MnO₂-MPs). In this study, we compared the accumulation of MnO₂-NPs and MnO₂-MPs in different tissues and evaluated their effects on mitochondrial complexes in isolated mitochondria. Our results showed that intravascular (iv) administration of the MnO₂-NPs in the same dose compared to the MnO₂-MPs resulted in more accumulation in the C57 mouse female tissues. The effect of MnO₂-NPs and MnO₂-MPs in mitochondria showed that complexes I and III play an important role in increasing ROS generation and this effect is related to type of tissue. Also, our results showed that exposure to MnO₂-NPs and MnO₂-MPs reduced the activity of mitochondrial complexes II and IV. Our results suggest that the toxicity of the MnO₂-NPs is higher than that of the MnO₂-MPs and can lead to the depletion of antioxidant status, likely induction of apoptosis, cancer, and neurodegenerative disease.

Abbreviations: NPs: nanoparticles; ROS: reactive oxygen species; SDH: succinate dehydrogenase; DCFH-DA: dichloro-dihydro-fluorescein diacetate; ELISA: enzyme-linked immunosorbent assay; MnO2-NPs: manganese oxide nanoparticles     

Continuous in vitro exposure of intestinal epithelial cells to E171 food additive causes oxidative stress,inducing oxidation of DNA bases but no endoplasmic reticulum stress

The whitening and opacifying properties of titanium dioxide (TiO₂) are commonly exploited when it is used as a food additive (E171). However, the safety of this additive can be questioned as TiO₂ nanoparticles (TiO₂-NPs) have been classed at potentially toxic. This study aimed to shed some light on the mechanisms behind the potential toxicity of E171 on epithelial intestinal cells, using two in vitro models: (i) a monoculture of differentiated Caco-2 cells and (ii) a coculture of Caco-2 with HT29-MTX mucus-secreting cells. Cells were exposed to E171 and two different types of TiO₂-NPs, either acutely (6–48?h) or repeatedly (three times a week for 3 weeks). Our results confirm that E171 damaged these cells, and that the main mechanism of toxicity was oxidation effects. Responses of the two models to E171 were similar, with a moderate, but significant, accumulation of reactive oxygen species, and concomitant downregulation of the expression of the antioxidant enzymes catalase, superoxide dismutase and glutathione reductase. Oxidative damage to DNA was detected in exposed cells, proving that E171 effectively induces oxidative stress; however, no endoplasmic reticulum stress was detected. E171 effects were less intense after acute exposure compared to repeated exposure, which correlated with higher Ti accumulation. The effects were also more intense in cells exposed to E171 than in cells exposed to TiO₂-NPs. Taken together, these data show that E171 induces only moderate toxicity in epithelial intestinal cells, via oxidation.     

Evaluation of cytotoxic,genotoxic and inflammatory response in human alveolar and bronchial epithelial cells exposed to titanium dioxide nanoparticles

The toxicity of titanium dioxide nanoparticles (TiO₂‐NPs), used in several applications, seems to be influenced by their specific physicochemical characteristics. Cyto‐genotoxic and inflammatory effects induced by a mixture of 79% anatase/21% rutile TiO₂‐NPs were investigated in human alveolar (A549) and bronchial (BEAS‐2B) cells exposed to 1–40 µg ml^–1 30 min, 2 and 24 h to assess potential pulmonary toxicity. The specific physicochemical properties such as crystallinity, NP size and shape, agglomerate size, surface charge and specific surface area (SSA) were analysed. Cytotoxic effects were studied by evaluating cell viability using the WST1 assay and membrane damage using LDH analysis. Direct/oxidative DNA damage was assessed by the Fpg‐comet assay and the inflammatory potential was evaluated as interleukin (IL)‐6, IL‐8 and tumour necrosis factor (TNF)‐α release by enzyme‐linked immunosorbant assay (ELISA). In A549 cells no significant viability reduction and moderate membrane damage, only at the highest concentration, were detected, whereas BEAS‐2B cells showed a significant viability reduction and early membrane damage starting from 10 µg ml^–1. Direct/oxidative DNA damage at 40 µg ml^–1 and increased IL‐6 release at 5 µg ml^–1 were found only in A549 cells after 2 h. The secretion of pro‐inflammatory cytokine IL‐6, involved in the early acute inflammatory response, and oxidative DNA damage indicate the promotion of early and transient oxidative‐inflammatory effects of tested TiO₂‐NPs on human alveolar cells. The findings show a higher susceptibility of normal bronchial cells to cytotoxic effects and higher responsiveness of transformed alveolar cells to genotoxic, oxidative and early inflammatory effects induced by tested TiO₂‐NPs. This different cell behaviour after TiO₂‐NPs exposure suggests the use of both cell lines and multiple end‐points to elucidate NP toxicity on the respiratory system. Copyright © 2014 John Wiley & Sons, Ltd.     

Tissue distribution and excretion of intravenously administered titanium dioxide nanoparticles

As the biosafety of nanotechnology becomes a growing concern, the in vivo nanotoxicity of nanoparticles (NPs) has been drawn an increasing attention. Titanium dioxide nanoparticles (TiO₂-NPs) have been developed for versatile use, but the pharmacokinetics of intravenously administered TiO₂-NPs have not been investigated extensively. In the present study, the rutile-type TiO₂-NPs with a size about 20 nm were labeled with CF680 and ¹²⁵I. The labeled TiO₂-NPs were injected in mice or rats with the concentration of 1 mg/ml and the dose of 10 mg/kg body weight and their tissue distribution and excretion were investigated by using ex vivo fluorescent imaging, γ-counter and TEM. The results indicated that the TiO₂-NPs mainly accumulated in liver and spleen and could be retained for over 30 days in these tissues due to the phagocytosis by macrophages. The excretion assay found that the excretory rate of TiO₂-NPs through urine was higher than that of feces, indicating that renal excretion was the main excretion pathway of TiO₂-NPs. Overall results of the present study provided important information on distribution and excretion of TiO₂-NPs in vivo, which would greatly promote the pharmacokinetics and in vivo nanotoxicity research of TiO₂-NPs.     

Amplification of arsenic genotoxicity by TiO2 nanoparticles in mammalian cells: new insights from physicochemical interactions and mitochondria

Titanium dioxide nanoparticles (TiO₂ NPs) have shown great adsorption capacity for arsenic (As); however, the potential impact of TiO₂ NPs on the behavior and toxic responses of As remains largely unexplored. In the present study, we focused on the physicochemical interaction between TiO₂ NPs and As(III) to clarify the underlying mechanisms involved in their synergistic genotoxic effect on mammalian cells. Our data showed that As(III) mainly interacted with TiO₂ NPs by competitively occupying the sites of hydroxyl groups on the surface of TiO₂ NP aggregates, resulting in more aggregation of TiO₂ NPs. Although TiO₂ NPs at concentrations used here had no cytotoxic or genotoxic effects on cells, they efficiently increased the genotoxicity of As(III) in human-hamster hybrid (A_L) cells. The synergistic genotoxicity of TiO₂ NPs and As(III) was partially inhibited by various endocytosis pathway inhibitors while it was completely blocked by an As(III)-specific chelator. Using a mitochondrial membrane potential fluorescence probe, a reactive oxygen species (ROS) probe together with mitochondrial DNA-depleted ρ⁰ A_L cells, we discovered that mitochondria were essential for mediating the synergistic DNA-damaging effects of TiO₂ NPs and As(III). These data provide novel mechanistic proof that TiO₂ NPs enhanced the genotoxicity of As(III) via physicochemical interactions, which were mediated by mitochondria-dependent ROS.     

Role of the crystalline form of titanium dioxide nanoparticles: Rutile,and not anatase,induces toxic effects in Balb/3T3 mouse fibroblasts

The wide use of titanium dioxide nanoparticles (TiO₂ NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO₂ NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO₂ NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO₂ NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO₂ NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials.In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO₂ NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size.     

Comparison of manganese oxide nanoparticles and manganese sulfate with regard to oxidative stress, uptake and apoptosis in alveolar epithelial cells

Due to their physicochemical characteristics, metal oxide nanoparticles (NPs) interact differently with cells compared to larger particles or soluble metals. Oxidative stress and cellular metal uptake were quantified in rat type II alveolar epithelial cells in culture exposed to three different NPs: manganese(II,III) oxide nanoparticles (Mn₃O₄-NPs), the soluble manganese sulfate (Mn-salt) at corresponding equivalent doses, titanium dioxide (TiO₂-NPs) and cerium dioxide nanoparticles (CeO₂-NPs). In the presence of reactive oxygen species an increased apoptosis rate was hypothesized. Oxidative stress was assessed by detection of fluorescently labeled reactive oxygen species and by measuring intracellular oxidized glutathione. Catalytic activity was determined by measuring catalyst-dependent oxidation of thiols (DTT-assay) in a cell free environment. Inductively coupled plasma mass spectrometry was used to quantify cellular metal uptake. Apoptosis rate was determined assessing the activity of caspase-3 and by fluorescence microscopic quantification of apoptotic nuclei. Reactive oxygen species were mainly generated in cells treated with Mn₃O₄-NPs. Only Mn₃O₄-NPs oxidized intracellular glutathione. Catalytic activity could be exclusively shown for Mn₃O₄-NPs. Cellular metal uptake was similar for all particles, whereas Mn-salt could hardly be detected within the cell. Apoptosis was induced by both, Mn₃O₄-NPs and Mn-salt. The combination of catalytic activity and capability of passing the cell membrane contributes to the toxicity of Mn₃O₄-NPs. Apoptosis of samples treated with Mn-salt is triggered by different, potentially extracellular mechanisms.     

Titanium dioxide nanoparticles induced intracellular calcium homeostasis modification in primary human keratinocytes. Towards an in vitro explanation of titanium dioxide nanoparticles toxicity

Abstract

Deciphering the molecular basis of toxicology mechanism induced by nanoparticles (NPs) remains an essential challenge. Ion Beam Analysis (IBA) was applied in combination with Transmission Electron Microscopy and Confocal Microscopy to analyze human keratinocytes exposed to TiO₂-NPs. Investigating chemical elemental distributions using IBA gives rise to a fine quantification of the TiO₂-NPs uptake within a cell and to the determination of the intracellular chemical modifications after TiO₂-NPs internalization. In addition, fluorescent dye-modified TiO₂-NPs have been synthesized to allow their detection, precise quantification and tracking in vitro. The internalization of these TiO₂-NPs altered the calcium homeostasis and induced a decrease in cell proliferation associated with an early keratinocyte differentiation, without any indication of cell death. Additionally, the relation between the surface chemistry of the TiO₂-NPs and their in vitro toxicity is clearly established and emphasizes the importance of the calcium homeostasis alteration in response to the presence of TiO₂-NPs.     

ROS-mediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells

Titanium dioxide nanoparticles (TiO₂ NPs) are among the top five NPs used in consumer products, paints and pharmaceutical preparations. Since, exposure to such nanoparticles is mainly through the skin and inhalation, the present study was conducted in the human epidermal cells (A431). A mild cytotoxic response of TiO₂ NPs was observed as evident by the MTT and NR uptake assays after 48 h of exposure. However, a statistically significant (p < 0.05) induction in the DNA damage was observed by the Fpg-modified Comet assay in cells exposed to 0.8 μg/ml TiO₂ NPs (2.20 ± 0.26 vs. control 1.24 ± 0.04) and higher concentrations for 6 h. A significant (p < 0.05) induction in micronucleus formation was also observed at the above concentration (14.67 ± 1.20 vs. control 9.33 ± 1.00). TiO₂ NPs elicited a significant (p < 0.05) reduction in glutathione (15.76%) with a concomitant increase in lipid hydroperoxide (60.51%; p < 0.05) and reactive oxygen species (ROS) generation (49.2%; p < 0.05) after 6 h exposure. Our data demonstrate that TiO₂ NPs have a mild cytotoxic potential. However, they induce ROS and oxidative stress leading to oxidative DNA damage and micronucleus formation, a probable mechanism of genotoxicity. This is perhaps the first study on human skin cells demonstrating the cytotoxic and genotoxic potential of TiO₂ NPs.     

DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells

Abstract

We investigated the genotoxic responses to two types of TiO₂ nanoparticles (<25 nm anatase: TiO₂-An, and <100 nm rutile: TiO₂-Ru) in human hepatoma HepG2 cells. Under the applied exposure conditions the particles were agglomerated or aggregated with the size of agglomerates and aggregates in the micrometer range, and were not cytotoxic. TiO₂-An, but not TiO₂-Ru, caused a persistent increase in DNA strand breaks (comet assay) and oxidized purines (Fpg-comet). TiO₂-An was a stronger inducer of intracellular reactive oxygen species (ROS) than TiO₂-Ru. Both types of TiO₂ nanoparticles transiently upregulated mRNA expression of p53 and its downstream regulated DNA damage responsive genes (mdm2, gadd45α, p21), providing additional evidence that TiO₂ nanoparticles are genotoxic. The observed differences in responses of HepG2 cells to exposure to anatase and rutile TiO₂ nanoparticles support the evidence that the toxic potential of TiO₂ nanoparticles varies not only with particle size but also with crystalline structure.     

Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro

With the extensive application of titanium dioxide (TiO₂) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO₂ NPs. The purpose of this study is to evaluate the genotoxicity of TiO₂ NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO₂ NPs (75 ± 15 nm) through intragastric administration at 0, 10, 50 and 200 mg/kg body weight every day for 30 days. The γ-H₂AX assay showed TiO₂ NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO₂ NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO₂ NPs at the dose of 0, 5, 10, 20, 50 and 100 μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24 h and 48 h exposure. Significant DNA damage was only observed at the concentration of 100 μg/mL after 24 h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100 μg/mL after 2 h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO₂ NPs, and TiO₂ NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO₂ NPs induced genotoxic effects.     

Nanoparticle size and combined toxicity of TiO2 and DSLS (surfactant) contribute to lysosomal responses in digestive cells of mussels exposed to TiO2 nanoparticles

The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO₂ nanoparticles (NPs) in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10?mg Ti/L either as TiO₂ NPs (60 and 180?nm) or bulk TiO₂, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO₂ NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO₂ NPs of 60?nm were internalised within digestive cell lysosomes to a higher extent than TiO₂ NPs of 180?nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO₂ NPs although more markedly after exposure to TiO₂-60 NPs. Exposure to bulk TiO₂ and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO₂ and DSLS.     

The size‐dependent genotoxic potentials of titanium dioxide nanoparticles to endothelial cells

Despite intensive research activities, there are still many major knowledge gaps over the potential adverse effects of titanium dioxide nanoparticles (TiO₂‐NPs), one of the most widely produced and used nanoparticles, on human cardiovascular health and the underlying mechanisms. In the present study, alkaline comet assay and cytokinesis‐block micronucleus test were employed to determine the genotoxic potentials of four sizes (100, 50, 30, and 10 nm) of anatase TiO₂‐NPs to human umbilical vein endothelial cells (HUVECs) in culture. Also, the intracellular redox statuses were explored through the measurement of the levels of reactive oxygen species (ROS) and reduced glutathione (GSH) with kits, respectively. Meanwhile, the protein levels of nuclear factor erythroid 2‐related factor 2 (Nrf2) were also detected by western blot. The results showed that at the exposed levels (1, 5, and 25 μg/mL), all the four sizes of TiO₂‐NPs could elicit an increase of both DNA damage and MN frequency in HUVECs in culture, with a positive dose‐dependent and negative size‐dependent effect relationship (T100 < T50 < T30 < T10). Also, increased levels of intracellular ROS, but decreased levels of GSH, were found in all the TiO₂‐NP‐treated groups. Intriguingly, a very similar manner of dose‐dependent and size‐dependent effect relationship was observed between the ROS test and both comet assay and MN test, but contrary to that of GSH assay. Correspondingly, the levels of Nrf2 protein were also elevated in the TiO₂‐NP‐exposed HUVECs, with an inversely size‐dependent effect relationship. These findings indicated that induction of oxidative stress and subsequent genotoxicity might be an important biological mechanism by which TiO₂‐NP exposure would cause detrimental effects to human cardiovascular health.     

Close association of intestinal autofluorescence with the formation of severe oxidative damage in intestine of nematodes chronically exposed to Al(2)O(3)-nanoparticle

In nematodes, acute exposure (24-h) to 8.1–30.6 mg/L Al₂O₃-nanoparticles (NPs) or Al₂O₃ did not influence intestinal autofluorescence, whereas chronic exposure (10-d) to Al₂O₃-NPs at concentrations of 8.1–30.6 mg/L or Al₂O₃ at concentrations of 23.1–30.6 mg/L induced significant increases of intestinal lipofuscin accumulation, and formation of severe stress response and oxidative damage in intestines. Moreover, significant differences of intestinal autofluorescence, stress response and oxidative damage in intestines of Al₂O₃-NPs exposed nematodes from those in Al₂O₃ exposed nematodes were detected at examined concentrations. Oxidative damage in intestine was significantly correlated with intestinal autofluorescence in exposed nematodes, and oxidative damage in intestine was more closely associated with intestinal autofluorescence in nematodes exposed to Al₂O₃-NPs than exposed to Al₂O₃. Thus, chronic exposure to Al₂O₃-NPs may cause adverse effects on intestinal lipofuscin accumulation by inducing the formation of more severe oxidative stress in intestines than exposure to Al₂O₃ in nematodes.     

Involvement of JNK and P53 activation in G2/M cell cycle arrest and apoptosis induced by titanium dioxide nanoparticles in neuron cells

Despite that applications of titanium dioxide nanoparticles (TiO₂-NPs) have been developed in the fields of paints, waste water treatment, sterilization, cosmetics, food additive, bio-medical ceramic and implant biomaterials and so on, relatively few studies have been conducted to determine the neurotoxicity of TiO₂-NPs exposure. In the present study, we investigated the cytotoxicity of TiO₂-NPs using PC12 cells and intended to clarify the molecular mechanisms underlying the biological effects of TiO₂-NPs. PC12 cell is a type of cells, which have been used as an in vitro model of dopaminergic neurons for neurodegenerative diseases research. In addition, the roles of the particle size and crystal structure of TiO₂-NPs to the neurotoxicity were also investigated. The anatase TiO₂-NPs displayed a dose-dependent behavior on decreasing cell viability, increasing levels of lactate dehydrogenase (LDH), activating oxidative stress, inducing apoptosis, disturbing cell cycle, triggering JNK- and p53-mediated signaling pathway. In comparison to anatase TiO₂-NPs, the rutile TiO₂-NPs showed moderately toxic effect on neuron cells. The micron-sized TiO₂ did not exhibit any toxic response. It is suggested from our results that reactive oxygen species (ROS) have a mediation effect to oxidative stress and up-regulation of JNK and P53 phosphorylation involved in mechanistic pathways of TiO₂-NPs can induce apoptosis and cell cycle arrest in PC12 cells. In addition, both the size and crystal structure of TiO₂-NPs exposure contributed to the neurotoxicity. Nanoparticles were more toxic than micrometer-sized particles and the anatase form were more toxic than the rutile.     

Titanium dioxide nanoparticles prime a specific activation state of macrophages

Titanium dioxide nanoparticles (TiO₂ NPs) are widely used in foods, cosmetics, and medicine. Although the inhalation toxicity of TiO₂ NPs has been studied, the potential adverse effects of oral exposure of low-dose TiO₂ NPs are largely unclear. Herein, with macrophage cell lines, primary cells, and mouse models, we show that TiO₂ NPs prime macrophages into a specific activation state characterized by excessive inflammation and suppressed innate immune function. After a month of dietary exposure in mice or exposure in vitro to TiO₂ NPs (10 and 50?nm), the expressions of pro-inflammatory genes in macrophages were increased, and the expressions of anti-inflammatory genes were decreased. In addition, for macrophages exposed to TiO₂ NPs in vitro and in vivo, their chemotactic, phagocytic, and bactericidal activities were lower. This imbalance in the immune system could enhance the susceptibility to infections. In mice, after a month of dietary exposure to low doses of TiO₂ NPs, an aggravated septic shock occurred in response to lipopolysaccharide challenge, leading to elevated levels of inflammatory cytokines in serum and reduced overall survival. Moreover, TLR4-deficient mice and primary macrophages, or TLR4-independent stimuli, showed less response to TiO₂ NPs. These results demonstrate that TiO₂ NPs induce an abnormal state of macrophages characterized by excessive inflammation and suppressed innate immune function in a TLR4-dependent manner, which may suggest a potential health risk, particularly for those with additional complications, such as bacterial infections.